PROJECT SUMMARY Clostridioides difficile is a mucosal-associated pathogen that can cause life-threatening illness. The bacterium is a leading source of hospital-acquired GI infections, but is emerging in the healthy population. Asymptomatic colonization is common and only leads to productive infection in some individuals, and a subset of these patients will have a relapsing, more severe disease. C. difficile secretes toxin B (TcdB), an inactivator of small GTPases that induces epithelial cell death. This toxin helps C. difficile establish the niche it needs for productive infection. C. difficile requires perturbation in the microbiome to initiate disease. Although recent studies have revealed some means of how it overcomes commensals, we still do not fully understand how C. difficile establishes infection or initiates re-infection. Host immune responses likely play a role. Our long-term goal is to identify how cytokine biology may be therapeutically targeted to alleviate initial C. difficile infection or relapses in high-risk patients. Group 3 innate lymphocytes (ILC3s) are rare immune cells often found in mucosal tissues. They produce high levels of IL-22, a critical modulator of mucosal tissue responses. IL-22 is important for maintaining intestinal homeostasis in health and disease. Through maintenance of the epithelial barrier, the cytokine is protective in GI infections, although the role of IL-22 in C. difficile infection is not clear. In our studies investigating interactions between toxins and ILC3s, we examined the effects of TcdB on IL-22. Our preliminary data show that TcdB induces IL-22 in ILC3s in a GTPase-dependent manner. Pharmacological inhibition suggests that upregulation of IL-22 is through Cdc42. These data form the premise for our hypothesis that C. difficile modulates the host immune response. The overall objective for this application is to understand signaling pathway(s) through which TcdB may modulate ILC3 production of IL-22 and ascertain the benefits of elevated IL-22 to C. difficile during infection. In Aim 1 we will examine how the small GTPase Cdc42 may regulate IL-22 production in activated ILC3s. Through genetic or siRNA-mediated deletion of Cdc42 in ILC3s, we will examine if this small GTPase is a negative regulator of ILC3 activation as well as examine if there are links between Cdc42 and other signaling pathways, including STAT3 and MAPKs. In Aim 2, we will examine the interactions between C. difficile and TcdB, and IL-22 and the GI tissues. We will examine the function of IL- 22 signaling in C. difficile infection using a reductionist approach with colonic organoids. Studies will examine if IL-22 protects against TcdB-mediated apoptosis and identify which IL-22-inducible factors contribute to altering the niche. An in vivo C. difficile infection model will complement in vitro experiments. This study may identify new signaling pathways involved in regulation of IL-22, which is of interest to both the infectious disease and chronic inflammation fields. We will also have a more complete understanding of how increased IL-22 levels modulate the inflammatory environment to favor C. difficile over other bacteria in the GI tract. This has implications on how IL-22 biology may be manipulated in preventing primary or relapsing C. difficile infection.